Nucleotide-Dependent Membrane Interaction and Dimerization of K-Ras4B

Abstract

Ras proteins are membrane-associated, small guanine nucleotide-binding GTPases that control cell survival and proliferation. They consist of highly homologous catalytic domains and flexible C-terminal hypervariable regions (HVRs) that differ significantly across Ras isoforms, H-Ras, N-Ras, K-Ras4A and K-Ras4B, where KRas4B is among the frequently mutated oncogenes in human tumors. Recent NMR experiments discovered that the HVR of GDP-bound K-Ras4B extensively interacts with the catalytic domain. However, the HVR weakly interacts with the catalytic domain when the protein is in the GTP-bound state. Molecular dynamics (MD) simulations confirmed tight interaction of HVR with catalytic domain in the GDP-bound state, but not GTP-bound K-Ras4B, suggesting that in the GDP-bound state, an HVR domain could adopt a β-strand conformation, extending the β-sheet in the active site of the catalytic domain. Here we modeled K-Ras4B membrane interaction and dimerization. Membrane binding of K-Ras4B through the anchoring of the positively charged HVR is critical to its function as an oncogene and initiates signaling events. Recent studies showed that post-translationally modified HVR peptide spontaneously inserts the farnesyl group into the zwitterionic (DOPC) and anionic (DOPC:DOPS=4:1) bilayers in the liquid phase, but not into the DPPC bilayer in the gel phase. Further, spontaneous membrane insertion of the farnesyl group in full-length K-Ras4B strongly depends on the nucleotide type. The HVR of K-Ras4B-GTP preferentially interacts with lipids through the farnesyl insertion, while the HVR of K-Ras4B-GDP rather binds the catalytic domain and inserts less frequently the farnesyl into the lipid bilayer. Remarkably, K-Ras4B-GTP, but not GDP-bound, is able to form stable homodimers with different dimer interfaces, suggesting that the nucleotide-dependent dimerization with various dimer interfaces can resolve nanoclustering and cluster reorganization accomplishment with Raf's activation. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E.